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Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2−expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

Rewarding experiences are often well remembered, and such memory formation is known to be dependent on dopamine modulation of the neural substrates engaged in learning and memory; however, it is unknown how and where in the brain dopamine signals bias episodic memory toward preceding rather than subsequent events. Here we found that photostimulation of channelrhodopsin-2-expressing dopaminergic fibers in the dentate gyrus induced a long-term depression of cortical inputs, diminished theta oscillations, and impaired subsequent contextual learning. Computational modeling based on this dopamine modulation indicated an asymmetric association of events occurring before and after reward in memory tasks. In subsequent behavioral experiments, preexposure to a natural reward suppressed hippocampus-dependent memory formation, with an effective time window consistent with the duration of dopamine-induced changes of dentate activity. Overall, our results suggest a mechanism by which dopamine enables the hippocampus to encode memory with reduced interference from subsequent experience.

The cortical cholinergic input system has been described as a neuromodulator system that influences broadly defined behavioral and brain states. The discovery of phasic, trial-based increases in extracellular choline (transients), resulting from the hydrolysis of newly released acetylcholine (ACh), in the cortex of animals reporting the presence of cues suggests that ACh may have a more specialized role in cognitive processes. Here we expressed channelrhodopsin or halorhodopsin in basal forebrain cholinergic neurons of mice with optic fibers directed into this region and prefrontal cortex. Cholinergic transients, evoked in accordance with photostimulation parameters determined in vivo, were generated in mice performing a task necessitating the reporting of cue and noncue events. Generating cholinergic transients in conjunction with cues enhanced cue detection rates. Moreover, generating transients in noncued trials, where cholinergic transients normally are not observed, increased the number of invalid claims for cues. Enhancing hits and generating false alarms both scaled with stimulation intensity. Suppression of endogenous cholinergic activity during cued trials reduced hit rates. Cholinergic transients may be essential for synchronizing cortical neuronal output driven by salient cues and executing cue-guided responses.

Auditory information is relayed from the cochlea along parallel pathways and reaches the inferior colliculus (IC) and the medial geniculate body (MGB) en route to the cortex. Although the ascending tectothalamic pathway to the ventral division of the MGB is regarded as a high-fidelity information-bearing channel, the roles of the pathways to the dorsal and medial divisions are more opaque. Here, we show fundamental differences between these ascending pathways using an in vitro slice preparation. Using photostimulation, we found three main patterns of input (excitatory, inhibitory, and mixed) that differed in each pathway. Furthermore, electrical stimulation of the central nucleus of the IC evoked a depressing response in the MGB with no metabotropic glutamate (mGlu) receptor component, whereas stimulation of the lateral cortex of the IC evoked a facilitating response with an mGlu receptor component. These data suggest that the ascending tectothalamic pathways are functionally distinct from one another.

We demonstrate that channelrhodopsin-2 (CR), a light-gated ion channel that is conventionally activated by using visible-light excitation, can also be activated by using IR two-photon excitation (TPE). An empirical estimate of CR's two-photon absorption cross-section at λ = 920 nm is presented, with a value (260 ± 20 GM) indicating that TPE stimulation of CR photocurrents is not typically limited by intrinsic molecular excitability [1 GM = 10 −50 (cm 4 s)/photon]. By using direct physiological measurements of CR photocurrents and a model of ground-state depletion, we evaluate how saturation of CR's current-conducting state influences the spatial resolution of focused TPE photostimulation, and how photocurrents stimulated by using low-power scanning TPE temporally summate. We show that TPE, like visible-light excitation, can be used to stimulate action potentials in cultured CR-expressing neurons.

Spatial vision in different organisms is mediated by 2 classes of photoreceptors: microvillar and ciliary. Recently, additional photosensitive cells implicated in nonvisual light-dependent functions have been identified in the mammalian retina. A previously undescribed photopigment, melanopsin, underlies these photoresponses, and it has been proposed that its transduction mechanisms may be akin to the lipid-signaling scheme of invertebrate microvillar receptors, rather than the cyclic-nucleotide cascade of vertebrates. Melanopsin has an ancient origin in deuterostomia, and expresses in 2 morphologically distinct classes of cells in the neural tube of Amphioxus, the most basal extant chordate: pigmented ocelli, and Joseph cells. However, to our knowledge, their physiology and alleged photosensitivity had never been investigated. We dissociated both types of cells, and conclusively demonstrated by patch-electrode recoding that they are primary photoreceptors; their receptor potential is depolarizing, accompanied by an increase in membrane conductance. The action spectrum peaks in the blue region, ≈470 nm, similar to the absorption of melanopsin in vitro. The light-dependent conductance rectifies inwardly; Na and Ca are differentially implicated in the 2 cell types. Fluorescence Ca imaging reveals that photostimulation rapidly mobilizes calcium from internal stores. Intracellular 1,2-bis(2-aminophenoxy)ethane- N , N , N ′, N ′-tetraacetate severely impairs the photoresponse, indicating that light-evoked Ca elevation is an important event in photoexcitation. These observations support the notion that the lineage of microvillar photoreceptors and its associated light-signaling pathway also evolved in the chordates. Thus, Joseph cells and pigmented ocelli of the Amphioxus may represent a link between ancestral rhabdomeric-like light sensors present in prebilaterians and the circadian photoreceptors of higher vertebrates.

An approach for high-spatial-resolution mapping of functional circuitry in living mammalian brain slices has been developed. The locations of neurons making functional synaptic connections to a single neuron are revealed by photostimulation of highly restricted areas of the slice (50-100 microns in diameter) while maintaining a whole-cell recording of the neuron of interest. Photostimulation is achieved by bathing brain slices in a molecularly caged form of the neurotransmitter glutamate [L-glutamic acid alpha-(4,5-dimethoxy-2-nitrobenzyl) ester], which is then converted to the active form by brief pulses (< 1 ms in duration) of ultraviolet irradiation. Direct activation of receptors on recorded neurons in rat hippocampus and ferret visual cortex demonstrates that photostimulation is reliable and reproducible and can be repeated at the same site at least 30 times without obvious decrement in neuronal responsiveness. Photostimulation of presynaptic neurons at sites distant to the recorded neuron evoked synaptic responses in hippocampal and cortical cells at distances of up to several millimeters from the recorded neuron. Stimulation of 25-100 distinct presynaptic sites while recording from a single postsynaptic neuron was easily achieved. Caged glutamate-based photostimulation eliminates artifacts and limitations inherent in conventional stimulation methods, including stimulation of axons of passage, desensitization, and poor temporal resolution of "puffer" pipettes, and current artifacts of iontophoretic application. This approach allows detailed physiological investigation and manipulation of the complex intrinsic circuitry of the mammalian brain.